INTRODUCTION

Endocannabinoids (via cannabinoid CB1 receptoractivation) are physiological regulators of feeding behaviour (1),and CB1 receptors are widely expressed in the central andperipheral nervous systems (2). In the brain, CB1 receptors havebeen identified in pathways responsible for reward processes andfor energy balance (3). In the periphery, cannabinoid CB1receptors are expressed in several areas of the body includingfat, muscle, liver, and the digestive tract (4).

Further evidence that endogenous cannabinoids are involvedin central nervous system appetite regulation is derived fromobservations that the direct administration of anandamide intothe ventromedial hypothalamus stimulates food intake (5).Moreover, it has been observed that concentrations of 2-arachidonoyl glycerol in the limbic forebrain and hypothalamuscorrelate positively with the stimulation of food intake in rats(6). Low doses of the exogenous cannabinoid agonist THC areknown to induce hyperphagia in rats through the stimulation ofcannabinoid CB1 receptors (7). This action is blocked or

reversed by the selective CB1 receptor antagonists rimonabant(SR141716A) (8, 9) and AM 251 (10, 11). The appetitesuppressant effects of rimonabant have been observed after bothacute and chronic administration (12), and also for various dietsdiffering in palatability (13).

Several studies have suggested that the chronic administrationof cannabinoid receptor antagonists induces a decrease in the totalnumber of CB1 receptors within brain structures, including thelimbic system and the cerebral cortex (14). In addition, lowerlevels of mRNA (which codifies for cannabinoid CB1 receptor),have been observed in the forebrain region (15).

Cannabinoid CB1 receptors regulate feeding by interactingwith both peripheral sensory terminals and hypothalamic circuits(16, 17). At the level of the hypothalamus, they may interact withneuropeptide circuits involved in feeding regulation, includingneuropeptide Y, melanocortin, and orexins, which are a type ofneuropeptides earlier described as hypocretins (18). Orexin Aadministered to the lateral hypothalamus increases appetite in adose-dependent manner (19). CB1 and OX1 receptors areexpressed in similar brain regions, such as the lateral

JOURNAL OF PHYSIOLOGY AND PHARMACOLOGY 2015, 66, 2, 181-190www.jpp.krakow.pl

Original articles

I. MERROUN1, N. EL MLILI2, R. MARTINEZ1, J.M. PORRES1, J. LLOPIS1, H. AHABRACH3, P. ARANDA1, C. SANCHEZ GONZALEZ1, M. ERRAMI3, M. LOPEZ-JURADO1

INTERACTION BETWEEN OREXIN A AND CANNABINOID SYSTEM IN THE LATERALHYPOTHALAMUS OF RATS AND EFFECTS OF SUBCHRONIC INTRAPERITONEAL

ADMINISTRATION OF CANNABINOID RECEPTOR INVERSE AGONISTON FOOD INTAKE AND THE NUTRITIVE UTILIZATION OF PROTEIN

1Department of Physiology, School of Pharmacy, University of Granada, Granada, Spain; 2Institut de Formation aux Carrieres de Sante, Rabat, Morocco; 3Department of Biology, School of Sciences, Abdelmalek Essaadi University, Tetuan, Morocco

Crosstalk may occur between cannabinoids and other systems controlling appetite, since cannabinoid receptors are presentin hypothalamic circuits involved in feeding regulation, and likely to interact with orexin. In this study, animmunohistochemical approach was used to examine the effect of the intracerebroventricular administration of cannabinoidreceptor inverse agonist AM 251 on orexin neuropeptide in the hypothalamic system. AM-activated neurons were identifiedusing c-Fos as a marker of neuronal activity. The results obtained show that AM 251 decreases orexin A immunoreactivity,and that it increases c-Fos-immunoreactive neurons within the hypothalamus when compared with the vehicle-injectedcontrol group. We also studied the effects of subchronic intraperitoneal administration of AM 251 on food intake, bodyweight, and protein utilization. The administration of AM 251 at 1, 2, or 5 mg/kg led to a significant reduction in foodintake, along with a significant decrease in the digestive utilization of protein in the groups injected with 1 and 2 mg/kg.There was a dose-related slowdown in weight gain, especially at the doses of 2 and 5 mg/kg, during the initial days of thetrial. The absence of this effect in the pair-fed group reveals that any impairment to digestibility was the result ofadministering AM 251. These data support our conclusion that hypothalamic orexigenic neuropeptides are involved in thereduction of appetite and mediated by the cannabinoid receptor inverse agonist. Furthermore, the subchronic administrationof AM 251, in addition to its effect on food intake, has significant effects on the digestive utilization of protein.

K e y w o r d s : cannabinoid, food intake, body weight, protein digestibility, c-Fos expression, orexin A, immunoreactivity,cannabinoid CB1 receptor agonist

hypothalamus (20, 21). Therefore, crosstalk between orexin Aand the cannabinoid may occur within the brain since both areco-expressed in the same regions as the hypothalamic neuronsinvolved in feeding behavior and energy homeostasis (22-24).

The presence of CB1 receptors in the nerve terminals thatinnervate the gastrointestinal tract (25) suggests that they mightnot only affect daily food intake, but also nutrient utilization atthe digestive and metabolic level. The present study reports noveldata regarding the effect of cannabinoids on protein utilization.

Following previous research showing that CB1 receptoragonists enhance feeding behaviour (26) and that CB1 receptorantagonists reduce food intake, the aim of the present study wasto examine the effects that the subchronic administration of thecannabinoid receptor inverse agonist AM 251 at different doseswould have on food intake, body weight, and protein utilizationby rats. In order to identify the hypothalamic circuits implicatedin these effects, c-Fos expression was used to determineneuronal activity at these levels. In addition, potentialinteractions between AM 251 and orexin A were examined.

MATERIALS AND METHODS

Animals

Male Wistar rats with an initial body weight of 150 ± 20 gwere housed in individual cages designed for the separatecollection of faeces and urine. The cages were located in a wellventilated thermostatically controlled room (21 ± 1°C, 12 hlight/dark period) and the animals were given a standard ratchow and water ad libitum. All experiments were undertakenaccording to Directional Guides Related to Animal Housing andCare. The European guidelines for the care and use of animalswere followed and all experimental procedures involvinganimals were approved by the Ethics Committee for AnimalExperimentation of the University of Granada (Spain).

Drugs

The CB1 receptor inverse agonist, N-(piperidin-1-yl)-5 (4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (AM 251) (Tocris Cokson, Bristol, UK) wasdissolved in vehicle (Tween-80: dimethyl sulfoxide (DMSO):0.9% NaCl in 1:2:97 ratio.

Experimental groups

In order to study the effect of the subchronic (i.p.)administration of AM 251 on food intake, body weight andnutritive utilization, 40 male Wistar rats (n=10 animals per group)were distributed among the following experimental groups:

1 - Vehicle-injected control group2 - Group injected with AM 251 at dose of 1 mg/kg3 - Group injected with AM 251 at dose of 2 mg/kg4 - Group injected with AM 251 at dose of 5 mg/kg

A fifth pair-fed experimental group was included after theprevious four groups were completed to separate drug effects tothose related to differences in food intake. This fifthexperimental group was vehicle-injected and pair-fed to theamount of food consumed by the group injected with AM 251 atthe intermediate dose of 2 mg/kg.

For the immunohistochemistry study, four groups of maleWistar rats (n=5 animals per group) were used. In two groups,we studied the effect of the intracerebroventricularadministration of AM 251 on orexin expression, as follows:

1 - Vehicle-injected control group, reflecting orexin A expressionin the hypothalamic neurons.

2 - Experimental group injected with AM 251 at a dose of 1 µg/5 µl.In the other two groups, we studied the effect of the

intracerebroventricular administration of AM 251 on c-Fosexpression, as follows:1 - Vehicle-injected control group2 - Experimental group injected with AM 251 at a dose of

1 µg/5 µl.

Food intake and nutritive utilization studies

The effects of the subchronic (i.p.) administration, for eightdays, of CB1 receptor inverse agonist on food intake wereanalysed in presatiated rats as follows: the animals were alloweda period of ten days to adapt to the experimental conditions anddiet. Then, after 24 hours fasting a presatiation procedure wasapplied, in which the rats were allowed to eat the experimentaldiet ad libitum for 1 hour before drug administration to permit astandardised observation of the drug-induced feeding effect (27).After this initial presatiation period, the drugs were administereddaily (i.p.) during eight days. Three different fresh solutions ofdrug + vehicle were prepared daily. The solutions were preparedfor 0.4, 0.8 and 2 mg/mL concentrations by diluting AM 251 invehicle which was a mixture of Tween-80: dimethyl sulfoxide(DMSO): 0.9% NaCl 100 (1:2:97 ratio). Animals were dailyweighted and solution volume for each animal was thencalculated according to their weight to get a final concentration of1, 2 and 5 mg/kg body weight following the protocol previouslyreported by Chambers et al. (28). Administration was performedin darkness. Thirty minutes after drug or vehicle administration,the rats were allowed free access to food. A previous study (29)examined the effect of acute administration, and the present studyconsiders the effect of subchronic administration, using the samedoses. Throughout the experimental period, the animals' bodyweight and food consumption were recorded daily 24 hours afterthe injection of vehicle or drugs, at 10 a.m. Faeces and urine werecollected daily for each rat, frozen at –20°C, weighed and groundfor analysis of protein content.

Composition analysis of diet, faeces and urine

According to the manufacturer, the macronutrientcomposition of the standard diet used in the present study wasadequate to meet the nutrient requirements of the experimentalanimals for crude energy, total nitrogen and ash content. The dietcomposition was as follows: energy, 13.0 MJ/kg; nitrogen, 26.1g/kg; fat, 41 g/kg; carbohydrates, 690 g/kg; fibre, 45 g/kg; ash,60.8 g/kg. Moisture content was determined by drying toconstant weight in an oven at 105 ± 1°C. Total nitrogen (N) wasmeasured in diet, faeces and urine according to Kjeldahl'smethod in order to determine the intake and faecal and urinaryexcretion of nitrogen to assess the digestive and metabolicutilization of nitrogen (30). Crude protein was calculated asnitrogen × 6.25.

Biological indices

The following indices and parameters were determined foreach group according to the formulas given below: apparentdigestibility coefficient (ADC) (1), retention (balance) (2), andpercent nitrogen retention/nitrogen absorption (%R/A) (3):

(1) ADC = {(I - F) / I}×100(2) Balance = I - (F + U)(3) % R/A = { I - (F + U) / (I - F)}×100

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where I = intake, F = faecal excretion and U = urinaryexcretion.

Immunohistochemical study

The animals were anaesthetized with chloral hydrate (400mg/kg) and placed in a stereotaxic apparatus (Kopf Instruments,Tujunga, CA). A stainless-steel cannula guide was implantedinto the brain above the third ventricle, following the methoddescribed by Paxinos & Waston (31) (AP -0.72 mm, L 0.0 mm,DV -6.2). As described previously (29), a 10d post-surgicalrecovery period was allowed in order to stabilize food intakebefore the experimental period.

The effects of AM 251 on hypothalamic neuropeptideswere analysed in partially satiated rats; to this end, the ratswere deprived of food but not of water for 24 hours before thebeginning of the experiments. They were then given free accessto food for 1 hour (29). Infusions of 1 µg cannabinoid inverseagonist AM 251 solutions were made at a rate of 1 µl/min anda volume of 5 µl was injected into the third ventricle. Theinjector was maintained in place for 1 min to allow diffusion ofthe drug into the brain and to reduce backflow through thecannula track.

One hour after drug administration, animals wereanaesthetised and transcardially perfused through the aorta with50 ml saline followed by 300 ml of a fixative solution containing4% paraformaldehyde in 0.1 M-phosphate buffer (PB), pH 7·4.The brains were removed, postfixed for 8 hours in the samefixative solution, and then in 30% sucrose in PB for 48 hours at4°C. Transverse 40-µm sections were cut on a sliding microtomeand stored in protecting solution (30% ethylene glycol /30%glycerol in 0.1 M NaK2PO4) at –20°C until processing.

The expression of orexin-A and c-Fos within thehypothamalus (six sections per rat) was determined usingrabbit polyclonal antibodies raised against orexin-A protein(AbCam, Cambridge, UK) or c-Fos (Calbiochem, Darmstadt,Germany).

The brain sections were incubated for 24 hours at roomtemperature with the primary c-Fos or orexin A antiserum(1/10000; 1/1000). The sections were then incubated for 1 hourat room temperature with a biotinylated goat anti-rabbitantiserum (1/200) (Vector Laboratories, Paris, France); thesections were then rinsed in PBS and incubated for 1 hour atroom temperature with the standard avidin-biotin peroxidase(1/50) (Vector Laboratories). Peroxidase was revealed accordingto the method of Shu et al. (32), using diaminobenzidinetetrahydrochloride (Sigma-Aldrich, St Louis, MO) as achromogen. The reaction was stopped and the sections werewashed, mounted on polylysine coated slides using a 0.1M PBand then dehydrated and cover-slipped before examination in

microscope. The photomicrographs were taken on an OlympusBX-41 microscope (Olympus Inc., Hamburg, Germany) coupledto a Sony 3CCD video camera. Quantification of the number ofc-Fos or orexin A immunopositive cells was performed usingImage J software.

Statistical analysis

Statistical differences among groups for daily food intakeand weight changes were analysed by time-repeated ANOVAwith time, treatment and time × treatment interaction as themain effects. Pairwise comparisons were made among thedifferent experimental groups at each time points selected(days 1–8) (n=10). Differences in the digestive utilization ofprotein between the experimental group given 2 mg/kg of AM251 and the pair-fed vehicle-administered group (Fig. 3) wereevaluated by Student's t-test (n=10). Statistical differences inthe nutritive utilization of protein were evaluated by one-wayANOVA. Multiple mean comparisons were performed usingDuncan's test (Table 1) (n=10). Statistical analysis was appliedto the data using Statgraphic Statistical Graphics 2.1 SystemSoftware (Statistical Graphics Corporation, Rockville, MD).The level of significance was set at P <0.05. The results ofimmunostaining were expressed as mean values (n=5) ±standard errors and statistical significance were determinedusing Student's t-test. Differences with a P-value <0.05 wereconsidered significant.

RESULTS

Effect of the subchronic administration of AM 251 on foodintake and weight changes

Following the administration of AM 251, a reduction in foodintake was observed in animals injected with doses of 1, 2, and5 mg/kg, compared with the vehicle-injected control group (Fig.1). These differences persisted throughout the experimentalperiod. Significant differences in food intake among the differentdoses tested were observed during the first four days of drugadministration, but disappeared 24 hours after the fourthinjection. The AM 251-derived reduction in food intake waslinked to significantly lower (P <0.05) cumulative weightchanges during the experimental period (Fig. 2), which wereparticularly evident for the 2 and 5 mg/kg doses, compared withthe vehicle-injected control group (time × treatment interaction,P <0.0001). Furthermore, weight changes in the experimentalgroup receiving AM 251 at a dose of 2 mg/kg were smaller thanin pair-fed control animals.

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Vehicle 1 mg/kg 2 mg/kg 5 mg/kg Intake (g 100/g body weight) 9.68±0.18c 8.12±0.12b 8.37±0.19b 7.98±0.11a

N intake (mg/d) 509.2±10.0b 442.1±14.8a 451.6±14.1a 420.1±14.1a

Fecal N (mg/d) 126.3±5.33b 140.8±9.86c 124.9±5.08b 100.8±6.05a

Urinary N (mg/d) 136.3±9.0b 121.0±9.3a 111.9±9.3a 105.9±12.0a

Absorbed N (mg/d) 367.1±9.9b 309.7±15.0a 341.0±14.7a 329.2±16.0a

ADC (%) 74.4±0.89b 66.7±1.23a 70.9±1.18a 76.3±1.18b

Balance (mg/d) 230.8±11.0a 188.7±17.6a 229.1±17.7a 223.3±20.3a

(%) R/A 61.2±2.45a 60.1±3.6a 66.4±3.21a 67.1±4.04a

a,b,c, Mean values within a row with unlike superscript letters were significantly different (P <0.05).

Table 1. Effect of subchronic intraperitoneal administration of AM 251 at different doses on the nutritive utilization of protein. Dataare mean ± S.E.M. of n=10 animals. ADC, apparent digestibility coefficient, (%R/A) percent nitrogen retention/nitrogen absorption.

Effect of the subchronic administration of AM 251 on proteinutilization

Nitrogen intake was significantly lower in all AM 251-injected animals compared with the vehicle-injected control group(Table 1). Faecal N excretion was significantly increased by the 1 mg/kg dose, while it was significantly reduced by the 5 mg/kgdose. The digestive utilization of protein (expressed as ADC) wassignificantly lower in the groups injected with the 1 and 2 mg/kgdoses than in the control group, whereas no significant differenceswere found between these groups and the animals injected withthe 5 mg/kg dose. On the other hand, a progressive increase in thedigestive utilization of protein was observed with growing dosesof AM 251.

Regarding the metabolic utilization of protein, expressed asthe percentage of retained to absorbed N, no significantdifferences were observed after comparing the groupsadministered varying doses of AM 251 (Table 1) and the controlgroup.

Pair-fed experiment

Although the amount of N ingested was similar in thevehicle-injected and in the animals injected 2 mg/kg (Table 2), asignificantly lower digestive utilization of protein was observedfor the latter experimental group compared with the pair-fedcontrol group (Fig. 3).

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Fig. 1. Effect of subchronic administration of AM 251 at different doses on food intake (g/day). Arrows indicate where differenttreatments were given. Values are means and the standard errors are depicted by vertical bars (n=10). (a) vehicle vs. pair-fed, (b)vehicle vs. AM 251 (1 mg/kg), (c) vehicle vs. AM 251 (2 mg/kg), (d) vehicle vs. AM 251 (5 mg/kg), (e) pair-fed vs. AM 251 (5 mg/kg),(f) AM 251 (1 mg/kg) vs. AM 251 (5 mg/kg), (g) AM 251 (2 mg/kg) vs. AM 251 (5 mg/kg), P <0.05.

Pair fed AM 2 mg/ kg

Intake (g 100/g body weight) 8.26±0.31b 8.37±0.19b

N Intake (mg/d) 450.47a 451.6±14.1a

Fecal N (mg/d) 89.81±4.16a 124.9±5.08b

Urinary N (mg/d) 120.4±8.5a 111.9±9.3a

Absorbed N (mg/d) 360.66±4.16a 341.0±14.7a

ADC (%) 80.71±0.78b 70.9±1.18a

Balance (mg/d) 240.26±8.02a 229.1±17.7a

(%) R/A 66.68±2.33a 66.4±3.21a

a,b Mean values within a row with unlike superscript letters were significantly different (P <0.05).

Table 2. Effect of subchronic intraperitoneal administration of AM 251 (2 mg/kg) on the nutritive utilization of protein. Data are mean± S.E.M. of n=10 animals. ADC, apparent digestibility coefficient, (%R/A) percent nitrogen retention/nitrogen absorption.

Effect of the intracerebroventricular administration of AM 251on c-Fos expression

Immunohistochemical analysis of the effects ofadministering AM 251 on neuronal activity at the lateral

hypothalamus showed that AM 251 significantly increased c-Fosexpression (1.5-fold, P <0.05), compared with the vehicle-injected control group (60.4 ± 4.0 and 40.4 ± 5.6, respectively;Fig. 4a and 4b).

Effect of the intracerebroventricular administration of AM 251on orexin A expression

Intracerebral injection of AM-251 induced a decrease inorexin expression in the lateral hypothalamus, compared withthe vehicle-injected control group (16.3 ± 1.0 and 46.4 ± 3.5,respectively P <0.01; Fig. 5a and 5b).

DISCUSSION

We studied the effects of the subchronic (i.p.) administrationof AM 251 at different doses, in order to better understand itspotential therapeutic action regarding the alteration of food intakeand of body weight. In addition to the effects observed on dailyfood intake and body weight, we provide data concerning theinfluence of the CB1 receptor inverse agonist on proteinutilization, together with data on the effect of theintracerebroventricular administration of AM 251 on c-Fos andorexin A expression in the lateral hypothalamus. The dataprovided complement those from prior studies conducted in ourlaboratory (29) concerning the effects of the acute administrationof this inverse agonist on food intake and related levels of 5-HT inthe ventromedial hypothalamus. In the present study, the

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Fig. 2. Effect of subchronic administration of AM 251 at different doses on body weight change (g). Values are means and the standarderrors are depicted by vertical bars (n=10). Changes are calculated taking the 24 h-fasted body weight as reference for all time points. (a)vehicle vs. pair-fed, (b) vehicle vs. AM 251 (2 mg/kg), (c) vehicle vs. AM 251 (5 mg/kg), (d) pair-fed vs. AM 251 (1 mg/kg), (e) pair-fedvs. AM 251 (2 mg/kg), (f) pair-fed vs. AM 251 (5 mg/kg), (g) AM 251 (1 mg/kg) vs. AM 251 (2 mg/kg), (h) AM 251 (1 mg/kg) vs. AM251 (5 mg/kg), P <0.05.

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Fig. 3. Effect of subchronic administration of AM 251 (2 mg/kg)on digestive utilization of protein expressed as apparentdigestibility coefficient (ADC). Values are means and thestandard errors are depicted by vertical bars (n=10).*Significantly different (P<0.05; Student’s t-test).

administration of AM 251 at 1, 2, or 5 mg/kg body weightproduced a significant reduction in food intake, accompanied by asignificant decrease in the digestive utilization of protein inanimals receiving 1 or 2 mg/kg of AM 251. These outcomesprovoked a dose-related slowdown of weight gain, especially atthe doses of 2 and 5 mg/kg during the initial days of the trial(observed with a dose of 2 mg/kg after the fourth injection andwith a dose of 5 mg/kg after the second one), which persisted untilthe end of the experiment. The fact that the accumulated weightchange was smaller in the group given AM 251 at a dose of 2mg/kg than in the pair-fed control group led us to consider that thiscannabinoid receptor inverse agonist could provoke effects otherthan those related to food intake, such as reducing absorption asmeasured by the coefficient of apparent digestibility (see below).Our results reflect a trend similar to that reported by Chambers etal. (11), and differences regarding methodological approach andexperimental design are probably responsible for the comparablysmaller effect observed. The reductions we measured in bodyweight could be related to changes in metabolism and/or increasedenergy expenditure, perhaps through the enhanced expression ofadiponectin gene, a circulating hormone that produces weight lossthrough the oxidation of free fatty acids, which in turn leads to thelipolysis of adipose tissue and to increased oxygen consumption(33, 34). The weight loss could also be attributed to reducedlocomotor activity caused by high doses of the drug. In someexperiments, the dose of 1.0 mg/kg of SR-141716A led tohypoactivity (35) and to diminished nicotine-induced hypoactivity

(36). In other studies, a single injection of AM 251 (0.25 mg/kg,i.p.) antagonized the motor stimulation induced by WIN 55,212-2(0.25 mg/kg, i.p.) in spontaneously hypertensive adolescent rats,suggesting that this effect was mediated by the cannabinoid CB1receptor, which is densely expressed in brain structures such as thecerebellum and the basal ganglia. These structures are known tomediate the initiation and coordination of movement (37). A highdensity of cannabinoid CB1 receptors in the axon terminals of thestriatal GABAergic neurons of the basal ganglia and of theglutamatergic granule cells of the cerebellum is probably involvedin motor control (38). Cannabinoid receptors may modulate bothinhibitory and excitatory neuronal transmission in the basalganglia, thus providing dual regulation of movement (39, 40).

The inhibitory effect of AM 251 on food intake observed inthe present study corroborates previous findings (10, 11, 28) inwhich varying doses and experimental periods of chronicadministration were used. Sustained levels of AM 251 and itsmetabolites in plasma could induce a strong inhibitor effect onthe hypothalamic nuclei involved in the regulation of food intakevia the CB1 receptors (41) located in the structures responsiblefor the control of energy balance and the incentive value of food,which are present in the central and peripheral nervous systems(27, 42, 43). The hypophagic effects of AM 251 are caused, atleast in part, by cannabinoid-induced alterations in bothexcitatory and inhibitory amino acid neurotransmission, and byconsequent changes in the activity of cells comprising thehypothalamic feeding circuitry, such as melanin-concentrating

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Fig. 4. Effect of intracerebroventricularinjection of AM 251 (1 ug/5 ul) onlateral hypothalamus c-Fos expression.(a) Values are means and the standarderrors are depicted by vertical bars(n=5; three sections counted per rat).*Significantly different (P<0.05,Student's t-test). (b) Photomicrographsshowing 50 um sections of the lateralhypothalamus immunostained for c-Fos. Rats treated with vehicle (V) andAM251 (AM).

hormone (44, 45), NPY (46), POMC (47), and orexins (48). Inaddition, hypothalamic levels of endogenous cannabinoids areinfluenced by feeding-relevant humoral factors such as leptin(49) and ghrelin (50).

The present study reports data supporting the notion thatthe endocannabinoid system may influence food intake byregulating the expression and/or action of the hypothalamicneuropeptide orexin, which is involved in feeding behaviourbut also takes part in the regulation of wakefulness affectingneuronal cellular morphology caused by GABAA receptordependent anaesthetics (51) and in protection of gastricmucosa (52). There is evidence that orexin A increases foodintake by delaying the onset of behaviourally normal satiety.The selective orexin receptor antagonist suppresses food intakeand advances the onset of the normal satiety sequence (48).According to Hilairet et al. (22), cannabinoid CB1 receptorsand orexin receptors are co-expressed by differenthypothalamic regions involved in feeding behaviour andenergy homeostasis, and their co-expression in structuresimplicated in feeding points out to the existence of aninteraction between cannabinoids and orexin, as supported byour data.

Regarding the central nervous system, theintracerebroventricular injection of the cannabinoid receptorinverse agonist AM 251 produced a significant increase in c-Fosexpression. This increase in neuronal activity was accompaniedby a significant decrease in the number of neurons expressingorexin A in the hypothalamus. These data are in agreement witha previous study showing that rimonabant produces a decrease inorexin expression and blocks the orexigenic effect of orexin A(53). Another study showed that the anorectic effect of AM 251-induced CB1 receptor blockade is due, at least in part, to the

inhibition of orexigenic peptide NPY production in thehypothalamus (54). Reduced orexin and NPY expression inanimals given AM 251 indicates a mutual interaction at the levelof the hypothalamic orexigenic neuropeptides. The hypophagiceffect of AM 251 might be mediated to some extent by orexinand NPY. It has also been shown (29) that one of theneurochemical mechanisms underlying the anorectic effect ofAM 251 could be related to its ability to differentially affect thefunctionality of serotonine-releasing neurons. Thus, AM 251seems to produce hypophagia by stimulating the mechanismsproducing the sensation of satiety.

The subchronic (i.p.) administration of AM 251 at doses of1 and 2 mg/kg produced significant hypophagia and reduceddigestive protein utilization. This reduced digestibility could bea result of either the reduced food intake or of theadministration of AM 251. Therefore, we conducted anadditional experiment in which a new group of animals waspair-fed to the experimental group, and given an intermediatedose of 2 mg/kg, thus avoiding the potential effect of the lowerfood intake. Although these two experimental groups had asimilar daily food intake, the digestive utilization of nitrogencontinued to be lower in the 2 mg/kg-injected animals than inthe pair-fed vehicle-injected group. Therefore, the decrease inthe digestive utilization of proteins was due to the effect of AM251 rather than to reduced food intake.

It has been reported that the activation of CB1 receptorsinhibit gastric motor function when given peripherically andprovide gastric mucosal protection (55) whereas CB1antagonists produce an increase in gastrointestinal motility (56,57), an effect that primarily involves a peripheral site of action(58). However, we cannot exclude that at least part of our resultsare mediated at a central level, via the diffusion of AM 251

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Fig. 5. Effect of intracerebroventricularinjection of AM 251 (1 ug/5 ul) onlateral hypothalamus orexin Aexpression. (a) Values are means andthe standard errors are depicted byvertical bars (n=5; six sections countedper rat). ** significantly different (P <0.01, Student's t-test). (b) Photomicrographs showing 50 umsections of the lateral hypothalamusimmunostained for orexin A. Ratstreated with vehicle (V) and AM251(AM).

(containing CB1 receptors) to the central nervous system. Theabove findings support our results concerning the significanteffect of AM 251 at the digestive level.

Regarding the experimental group given 5 mg/kg of AM251, we observed a significant reduction in faecal excretion, andtherefore a considerable increase in ADC. Greater digestiveutilization of N, expressed as ADC, could be a compensatoryeffect in response to the lower N intake, aimed at meetingnutrient requirements.

The i.p. administration of AM 251 led to a significantreduction in renal excretion of N, which was not dose-dependentand resulted in similar N balances among the 1 mg/kg, 2 mg/kg,5 mg/kg, and control animals. Nevertheless, no differences inurinary N excretion were found between the 2 mg/kg and thecontrol animals in the pair-fed experiment. This finding links theurinary excretion of N to the significant hypophagia induced byAM 251, rather than to any drug-related effect. This was to beexpected judging by the results of Deutsch et al. (59), whoreported the presence of CB1 receptors at the renal level. Inresponse to the lower daily intake of N, sufficient absorption ofthis nutrient took place to maintain the required levels of aminoacids. This compensatory effect was apparent in the similar Nbalance among all the animals injected with AM 251 (at 1, 2, and5 mg/kg) and the vehicle-injected control animals, as was a trendtoward higher indices of retained-to-absorbed N in the AM 251-injected animals, thus reflecting an acceptable degree ofutilization of absorbed N.

The results obtained lead us to conclude that there exists aninteraction between the hypothalamic orexin neuronal systemand the cannabinoid system. In addition to the hypophagic effectof AM 251, the subchronic administration of this inverse agonistalso produces significant effects on the digestive utilization ofprotein. This suggests that AM 251 could be applied to thetreatment of diseases caused by excessive food intake andimpaired energy metabolism. This is a novel finding, since forthe first time data are provided concerning the effect of CB1receptor inverse agonists on nutrient utilization for a particulardiet, beyond those related to daily food intake.

Acknowledgements: We thank Rosa Jiménez Llamas andEncarnación Rebollo for their skilful technical assistance. Thereis no conflict of interest that the authors should disclose. Thesources of funding were Projects AM34/04 and P07-AGR- 2704from Junta de Andalucía, Spain, and PROTARS III D14/47 fromCNRST, Morocco.

Conflict of interests: None declared.

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Received: August 19, 2014Accepted: January 7, 2015

Author's address: Prof. Maria Lopez Jurado, Department ofPhysiology, School of Pharmacy, University of Granada.Campus University of Granada s/n, Granada 18071, Spain.E-mail: mlopezj@ugr.es

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